Molecular and biochemical characterization of a novel cold-active and metal ion-tolerant GH10 xylanase from frozen soil

Haiyan Qiu,Zhongyuan Li,Hui Wang,Haiying Zhang,Shuang Li,Xuegang Luo,Yajian Song,Nan Wang,Hongpeng He,Hao Zhou,Wenjian Ma,Tongcun Zhang

doi:10.1080/13102818.2017.1359667

Abstract

ABSTRACTA novel xylanase gene of family 10 (xyn27) was obtained from the genomic DNA of frozen soil of Daxinganling in China by Touch-down polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL) PCR methods. Xyn27 contained a putative signal peptide (20 residues) and a catalytic motif of GH10 (327 residues) and shared the highest similarity (83%) with the reported GH10 xylanase (XM_003662144). The recombinant xyn27 was successfully expressed in Pichia pastoris GS115 and the optimal induction condition was 30 °C for 48 h. Xyn27 was demonstrated to be a cold-active xylanase, which shows its highest activity at 35 °C and still had 60.25%, 38.70% and 10.8% relative activity at 20 °C, 10 °C and 0 °C. Further analysis showed that Xyn27 has fewer arginine and more alanine residues compared with its mesophilic or thermophilic counterparts. The optimal pH of Xyn27 was 7.0, and it was stable after incubating under the pH range from 3.0 to 9.0 for 1 h. Besides, Xyn27 exhibited superior metal ion tolerance than other GH10 cold-active xylanases, being tolerant to most metal ions and organic solvents, and significantly enhanced by Ca2+, Mn2+ and Zn2+ metal ions. In addition, the Km, Vmax, kcat and kcat Km−1 against beechwood xylan were 13.42 mg mL−1, 9.07 μmol min−1 mg−1, 192.98 min−1 and 14.38 mL min−1 mg−1, and Xyn27 could completely degrade xylan into xylobiose. The features of cold activity and metal-ion tolerance suggested that xylanase Xyn27 could have potential application in basic research and various industries like food possessing.

Highlights

Hemicelluloses are the second most abundant plant polysaccharides in nature, which are attracting increasing attention due to their potential role as sustainable energy sources [1]
Based on the primary structure and amino-acid sequence, xylanases are classified into many glycoside hydrolase (GH) families, and xylanases belonging to GH10 and GH11 are mostly reported [3]
BlastP search indicated that Xyn27 belongs to GH family 10 and its amino acid sequence showed the highest similarity (83%) to the reported GH10 xylanase (XMç003662144) from Myceliophthora thermophila ATCC 42464

Summary

Introduction

Hemicelluloses are the second most abundant plant polysaccharides in nature, which are attracting increasing attention due to their potential role as sustainable energy sources [1]. Endo-1,4-b-D-xylanase (EC 3.2.1.8) plays a crucial role in the hydrolysis of the xylan back-bone by cleaving the b-1,4-glycosidic bond between xylose residues to release short xylooligosaccharides and xylose [1]. Cold-active xylanases are an exception, as they are most active at low and intermediate temperature, and thermolabile at high temperature, the use of cold-active xylanases at the industrial level could avoid alterations in the ingredient and product quality, but will reduce energy consumption and will avoid microbial development and fermentation [1,6].

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